This project is directed towards determining the structure of neuronal and glial cytoplasm, particularly as it pertains to axoplasmic transport, secretion, cell movement, and the organization of cell surface. Living cells or tissues are directly rapid-frozen and the structure of their cytoplasm is determined by one of two methods, freeze-etching or freeze-substitution. Axons in turtle optic nerves have different axoplasmic domains, each characterized by specific types of filaments and by its content of organelles. In other experiments, cultured myocytes grown on grids, frozen, and freeze-substituted are examined directly at high voltages in an electronmicroscope. Recently these cells have been examined in the living state at very high levels of resolution with a new video microscopic technique developed here; the same preparations are then frozen and examined in the electronmicroscope in order to correlate cytoplasmic movements and specific structural features seen in the living state with structures seen in more detail in the same cells in the whole mounts. So far, it has been shown that the cytoplasmic analog consists of fine filaments instead of a microtubular meshwork and that there are distinct cytoplasmic domains characterized by different amounts of Brownean movements and directed movements; positions within the cell; content of organelles; and content, types, and organization of filaments. Methods are being developed to enable immunocytochemical labels to be used to identify key structural components of the cytoplasm. We plan to use this direct method of observing the cytoskeleton to integrate and use these new concepts of cytoplasmic structure to develop a new understanding of the mechanism by which organelles move fast axoplasmic transport.